Date of Award

2015

Degree Type

Thesis

Degree Name

Doctor of Philosophy

School

School Of Medical Sciences

Faculty

Faculty of Health, Engineering and Science

First Advisor

Professor Mel Ziman

Second Advisor

Dr Elin Gray

Abstract

BACKGROUND: The incidence of skin cancer in Australia has increased rapidly in the last few decades. Ultraviolet radiation (UV) is a major risk factor for skin carcinogenesis. UV, particularly the UVB spectrum, causes formation of cyclobutane pyrimidine dimers (CPD) in cellular DNA. Persistent and incorrectly repaired CPDs lead to DNA mutations and consequently, formation of cutaneous lesions. Interestingly, recent epidemiological studies have shown a significant increase in skin cancer incidence in geographical locations with high environmental temperatures. Thus, heat stress may potentiate the effects of UV exposure and act as an additional risk factor for skin cancer. Previous studies in mice have shown that repeated and concurrent exposure to UVB and heat stress, increases the rate and incidence of cutaneous tumour formation relative to UVB alone. However, the effects of UVB plus heat on human epidermal cells have yet to be determined. Furthermore, the exact mechanisms responsible for the observed effects of heat stress need to be characterised in skin keratinocytes to increase knowledge of its risk in skin cancer.

Heat stress induces upregulation of heat shock proteins (HSPs), particularly HSP72 and HSP90 which are known to affect the activity of the p53 protein. Furthermore, heat stress has been linked with increased Sirtuin1 (SIRT1) protein activity. SIRT1 is an important histone deacetylase that helps maintain chromosomal integrity but can also induce post-translational modifications of the p53 protein. By mediating deacetylation of the p53 protein, SIRT1 can diminish the ability of p53 to bind to its downstream gene targets. The p53 protein is an integral mediator of the cellular stress response in skin cells, particularly keratinocytes. Thus, impairment of p53 transcription factor functions could compromise the ability of epidermal cells to mount an appropriate response to DNA damage. Moreover, loss of p53 function may induce survival of cells harbouring DNA lesions.

We hypothesise, therefore, that exposure to UVB plus heat induces survival of DNA damaged keratinocytes and that these cells escape apoptosis surveillance as a result of heat-mediated alteration to the p53 signalling pathway. Thus, exposure to heat stress could exacerbate the carcinogenic effects of UV and increase the risk of skin tumour formation in humans

AIMS: In this study, we aimed to determine whether repeated exposure to UVB followed immediately by heat stress (39°C) has a more damaging effect on human keratinocytes than UVB alone. In particular, we assessed the effects on DNA damage, apoptosis, cell cycle and DNA repair. Furthermore, we aimed to unravel the mechanism through which heat mediates the survival of UVB DNA-damaged keratinocytes, focusing on the effects on the p53 signalling pathway.

MATERIALS AND METHODOLOGY: Primary adult human epidermal keratinocytes (NHEK) and ex vivo punch biopsies of normal human skin called NativeSkin® (Genoskin, France), were used as experimental models for this study. A UV cabinet fitted with a TLUVB Narrowband lamp (Philips, GERMANY), with a spectral output of 290 -315 nm, was used to administer UVB irradiation at a dose of 1 KJ/m². Heat stress involved culture in a normal CO2 incubator, with temperature maintained at 39°C for three hours. The temperature used in the experiments was based on previous measurements of skin surface temperature of open cut miners, who are prone to intense heat stress, in the Pilbara region of Western Australia. For UVB plus heat exposures, cells and skin models were sequentially exposed to 1 KJ/m2 of UVB, (at room temperature), followed immediately by 3 hours incubation at 39°C once per day, for four consecutive days. Unexposed skin models and NHEK, maintained at 37ºC, were used as experimental controls. Cell proliferation, apoptosis and whole genome expression profiles were analysed at four hours post day 4 exposure, to understand earlier events, and at 2 days post-exposure, to assess persistent outcomes of these exposures.

Treated primary NHEK cells were counted in a Vi-CellTM Viability Analyser and the level of apoptosis for exposed primary cells was determined using Annexin V/Propidium Iodide apoptosis assay at 4 hours and 2 days post exposure. To determine the presence of DNA damage, total and active p53 protein, as well as total and active SIRT protein, in the skin models and primary NHEKs, immunohistochemistry and/or immunocytochemistry was performed. Skin FFPE and primary NHEKs were incubated with antibodies to thymine dimers (CPD, DNA damage) and p53 (total), acetylated p53-382 (active), SIRT1 (total) or SIRT1-p (active) antibodies.

To measure apoptosis in skin, an anti-pan-cytokeratin marker was used to label keratinocytes and active-caspase-3 antibodies were used to identify apoptotic cells. To determine the expression of p53-downstream target genes at 4 hours, quantitative RT-PCR was performed using TaqMan probes for BAX, Survivin (BIRC5), ERCC1 and XPC genes, with Human 18S gene as the endogenous reference gene. Relative quantification of the expression levels of each transcript in each sample were calculated using the Delta-Delta CT method relative to untreated controls. A whole genome expression analysis was performed at 2 days postexposure using the Human HT-12 Expression v4 BeadChip (Illumina, USA). The Ingenuity Pathway Analysis (IPA) (Qiagen, USA) software was used to annotate the effects of altered gene expression on cell function and upstream signalling pathways. Two-way ANOVA was used to analyse differences across treatment groups, while parametric unpaired t-tests were used to detect differences between specific treatment groups in all experimental categories, i.e. proliferation, apoptosis and gene expression, with p-values

RESULTS:

Outcome 1 Using ex vivo skin models and NHEKs, we show for the first time that UVB plus heat treated keratinocytes exhibit DNA damage, as observed after UVB treatment alone. However, apoptosis was significantly reduced, possibly as a result of inactivation of the p53-mediated stress response, in DNA damaged cells of UVB plus heat treated samples. Furthermore, whole genome expression and IPA upstream analysis showed that heat induces SIRT1 activation, which was confirmed via immunohistochemistry assays. Heat-induced SIRT1 expression was linked to a decrease in acetylated p53 and consequently, downregulation of p53-regulated pro-apoptotic and DNA damage repair genes. These results suggest that p53-mediated cell cycle arrest and apoptosis, known to be induced by UVB, are ablated with the addition of heat, leading to survival of DNA damaged cells after UVB plus heat treatment.

Outcome 2 – We further confirmed that SIRT1 activation did not inhibit the transcription of the p53 protein but mediated deacetylation of p53, resulting in significant deregulation of expression of p53 downstream gene targets and decreased keratinocyte apoptosis in UVB plus heat treated samples. Importantly, chemical inhibition of SIRT1 by Ex-527, a known chemical inhibitor of SIRT1, in UVB plus heat exposed keratinocytes, resulted in reactivation of the p53 signalling pathway and increased apoptosis of DNA damaged keratinocytes. This clearly demonstrated the role of heat-mediated SIRT1 activation in the survival of DNA damaged keratinocytes after exposure to UVB plus heat.

CONCLUSION: In this study, we showed that the efficiency of cellular stress response to UVB-induced DNA damage is diminished in the presence of heat and, for the first time,

provide a molecular mechanism that explains these effects. With the novel use of an ex vivo human skin model, this study showed that heat stress prevents human keratinocytes, damaged by UV irradiation, from undergoing apoptosis and/or necrosis. We found UV plus heat exposure mediates SIRT1 activation which has been found to induce deacetylation of p53 and, consequently, the inactivation of the p53 signalling pathway. SIRT1 inhibition precluded the downregulation of p53 signalling by UV plus heat exposure, restoring apoptosis levels to those observed in UVB-only exposures. Thus, we demonstrated that SIRT1 activation is the main molecular mechanism driving UVB plus heat-induced survival of DNA damaged keratinocytes.

Overall, the results of this study suggest that by allowing the survival of DNA damaged keratinocytes, via induction of SIRT1 activation, heat stress can exacerbate the carcinogenic effects of UVB radiation. Exposure to heat stress, in addition to UV, could therefore increase the accumulation of mutations in keratinocytes, possibly leading to the transformation of normal cells into pre-cancerous cells. Further research is warranted to determine the role of UVB plus heat in skin cancer pathogenesis. Such knowledge could be utilised in public health campaigns to decrease risk, particularly for people exposed to combinations of these environmental hazards in workplaces such as in the mining, construction and petroleum industries.

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